The specific spatio-temporal regulation of the 22 identified keratins during mammalian development led to the hypothesis that keratin filaments play a fundamental role in the organization and functional activities of embryonic and adult epithelia. The mouse keratin 8 (mK8) and 18 (mK18) form the first intermediate filament (IF) observed during embryogenesis in the trophectoderm of 3.5 days blastocysts. They are found later in most simple epithelia such as gut, lung, liver, breast and uterus. In carcinomas, their expression is conserved, and consequently anti-keratin antibodies have been used extensively for tumor typing. We introduced a targeted mK8 null mutation in the germ line of mice and found that mK8 deficiency leads to 90% embryonic lethality at 12 to 13 days of development. For the first time, we have a genetic mutant to ask specific questions about the mK8 function(s). The present proposal address fundamental questions about the normal and perhaps pathological function of simple epithelium keratin filaments, with some insights on mK8 gene regulation: 1) The mechanisms by which mK8 deficiency leads to embryonic lethality. 2) The functional significance of conserved "end" domains. 3) The functional significance of intermediate filament diversity. 4) The study of mK8 gene regulatory elements within the context of the multigene keratin locus A set of functional and ultrastructural studies of mK8- embryonic epithelia, and rescue experiments of mK8- embryos with variant forms of mK8, and other IF family members, will be performed for these purposes. Furthermore, chimeras resulting from the aggregation of wild type and mutant embryos will compare directly the ability of wild type and mutant cells to migrate and colonize diverse epithelia in a "competitive" environment. In addition, we propose to introduce a mK8 dominant-negative mutation and regulatory element deletions by gene targeting. This strategy will be most useful to alleviate the unspecific effects normally encountered with standard transgenic experiments. The effect of these targeted mutations will be evaluated during the differentiation of ES cells in vitro and in vivo directly in chimeras without the requirement for the time-consuming germ-line transmission. The rescue experiments and the subtle targeted mutations will define the best strategies to increase the variety of mutations that can be produced in the mouse genome.